GSM7011354: ChIP_Input; Plasmodium falciparum; ChIP-Seq
Parasites were resuspended in ice cold SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) supplemented with PMSF (100μM/ml) and protease inhibitor cocktail for 10 minutes on ice. Parasite lysate was then sonicated to generate the sheared chromatin of ~500bp and centrifuged at 1000g for 15 minutes. After the removal of debris from the lysate, the lysate was further diluted 10 times in ChIP dilution buffer (0.01% SDS, 0.1% Triton-X 100, 1.2mM EDTA, 16.7mM Tris, pH 8.1, and 150mM NaCl). Subsequently, the parasite lysates were precleared using protein A sepharose beads at 4°C for 1 hour. Cleared chromatins were incubated with pre-immune sera and immune MCM6 sera for overnight at 4°C on rotating wheel. ChIP and Control DNA libraries were generated using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (#E7645S). The method involves the end repair and A tailing and adapter ligation. The adapter-ligated DNA was enriched by PCR amplification by using Illumina index primers. The final amplified product were purified using Ampure beads to remove unused primers. The library was quantitated using Qubit DNA High Sensitivity quantitation assay and library quality was checked on Bioanalyzer 2100 using Agilent High Sensitivity DNA Kit. The librares were sequenced to generate 150 bases paired end reads using the use of HiSeq.